Sex-specific metabolic adaptations in transgenic mice overexpressing cytochrome b5 reductase-3.

Cytochrome b5 reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD+, and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females.

Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q10, but due to its highly lipophilic nature, Q10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney.

Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice.

Calorie restriction (CR) is the most robust non-genetic intervention to delay aging. However, there are a number of emerging experimental variables that alter CR responses. We investigated the role of sex, strain, and level of CR on health and survival in mice. CR did not always correlate with lifespan extension, although it consistently improved health across strains and sexes. Transcriptional and metabolomics changes driven by CR in liver indicated anaplerotic filling of the Krebs cycle together with fatty acid fueling of mitochondria. CR prevented age-associated decline in the liver proteostasis network while increasing mitochondrial number, preserving mitochondrial ultrastructure and function with age. Abrogation of mitochondrial function negated life-prolonging effects of CR in yeast and worms. Our data illustrate the complexity of CR in the context of aging, with a clear separation of outcomes related to health and survival, highlighting complexities of translation of CR into human interventions.

Mitochondrial ultrastructure and markers of dynamics in hepatocytes from aged, calorie restricted mice fed with different dietary fats.

In this paper we analyzed changes in hepatocyte mitochondrial mass and ultrastructure as well as in mitochondrial markers of fission/fusion and biogenesis in mice subjected to 40% calorie restriction (CR) for 18 months versus ad libitum-fed controls. Animals subjected to CR were separated into three groups with different dietary fats: soybean oil (also in controls), fish oil and lard. Therefore, the effect of the dietary fat under CR was studied as well. Our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. Finally, CR had no remarkable effects on the expression levels of fission and fusion protein markers. However, considerable differences in many of these parameters were found when comparing the CR groups, supporting the idea that dietary fat plays a relevant role in the modulation of CR effects in aged mice.

Differential regulation of hepatic apoptotic pathways by dietary olive and sunflower oils in the aging rat.

In this work we have studied how dietary fat affects aging-related changes in a number of factors that regulate rat hepatic apoptosis. Animals were fed lifelong with two experimental diets containing either virgin olive oil or sunflower oil as dietary fat. Caspases of the intrinsic and extrinsic pathways of apoptosis, Bcl-2 and Bax polypeptide levels, and plasma membrane neutral sphingomyelinase activity were determined at 6, 12, and 24 months of age. Caspase-8/10 activity (a marker of the extrinsic pathway) was not affected by either aging or dietary fat, but activities of both caspase-9 (a marker of the intrinsic pathway) and caspase-3 (an executioner caspase) were significantly depressed in liver from animals fed on a sunflower oil-based diet. These decreases were not observed in animals fed with a diet based on virgin olive oil, which also resulted in significantly lower Bcl-2/Bax ratios. On the other hand, in comparison with sunflower, dietary olive oil decreased oxidative stress in liver from aged rats, resulting in lower levels of membrane hydroperoxides and higher coenzyme Q levels in plasma membrane. Plasma membrane Mg(2+)-dependent neutral sphingomyelinase was strongly activated in aged rats fed on the sunflower oil diet, but no aging-related increase was observed in animals fed on the olive oil diet. Our results support that dietary oil can alter significantly the susceptibility of hepatocytes to different apoptotic stimuli by altering both pro- and anti-apoptotic mediators, which reinforces the importance of the diet in aging studies. Because virgin olive oil may increase susceptibility of hepatocytes to apoptosis induced through the intrinsic pathway under conditions of decreased oxidative stress, our results may have important implications to understand the potential beneficial effects of that edible oil against liver carcinogenesis during aging.

Stimulation of polyprenyl 4-hydroxybenzoate transferase activity by sodium cholate and 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate.

Polyprenyl 4-hydroxybenzoate transferase (Coq2p) plays a central role in ubiquinone biosynthesis. Coq2p mediates the conjugation of 4-hydroxybenzoate, the benzoquinone ring precursor, with the completed side chain. The activity is most easily assayed by measuring the rate of incorporation of 4-hydroxybenzoate as radiolabeled substrate into polyprenyl 4-hydroxybenzoate. The in vitro assay requires addition of a detergent into the reaction mixture to activate enzyme activity, and Triton X-100 is used for this purpose in the routine assay. We have found that both 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate and sodium cholate, but not sodium deoxycholate, lysophosphatidyl choline, or octylglucoside, significantly stimulate the activity over that measured with Triton X-100. High-performance liquid chromatography analysis of lipid extracts revealed that the increase of specific activity resulted in a similar increase in reaction product, this effect is due not merely to a better lipid extraction but also to the actual stimulation of enzyme activity. With our improved method, we were able to measure Coq2p activity with much greater sensitivity in both fresh and frozen/thawed mitochondria and in crude homogenates obtained from cultured cells. Our method will simplify evaluation of Coq2p activity in scarce biological materials, such as cells obtained from human tissue biopsies, and thus it will facilitate the biochemical characterization of ubiquinone deficiencies.

Enhanced anti-oxidant protection of liver membranes in long-lived rats fed on a coenzyme Q10-supplemented diet.

Coenzyme Q10 supplementation increases life-span of rats fed on a diet enriched with polyunsaturated fatty acids (Quiles, J.L., Ochoa, J.J., Huertas, J.R., Mataix, J., 2004b. Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet. Exp. Gerontol. 39, 189-194). Our study was set as a first attempt to establish a mechanistic link between life span extension and CoQ10 supplementation. When rats were fed on a PUFAn-6 plus CoQ10 diet, levels of CoQ10 were increased in plasma membrane at every time point compared to control rats fed on a PUFAn-6-alone diet. Ratios of CoQ9 to CoQ10 were significantly lower at every time point in both liver plasma membranes and homogenates of CoQ10-supplemented animals. CoQ10 supplementation did not affect cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1), which increased significantly with aging, but plasma membrane-bound NQO1 decreased significantly in the CoQ10-supplemented group at 12 months, when maximal incorporation of exogenous CoQ10 was observed. Neither aging nor the diet affected NADH-cytochrome b5 reductase levels. Glutathione-dependent anti-oxidant activities such as cytosolic glutathione-S-transferase (GST) and microsomal Se-independent glutathione peroxidase decreased with aging and supplementation with CoQ10 attenuated this decay. 2,2' Azobis amidinopropane (AAPH)-induced oxidation of membranes was significantly higher in aged rats, and supplementation with CoQ10 also inhibited this increase. Consistent with higher CoQ10 levels and enhanced anti-oxidant protection, plasma membrane Mg2+-dependent neutral sphingomyelinase was inhibited by dietary CoQ10 in aged rats. Our results support the involvement of thiol-dependent mechanisms in the potentiation of the anti-oxidant capacity of membranes in CoQ10-supplemented rats, further supporting the potentially beneficial anti-oxidative role of dietary CoQ10 during aging. The possibility that a decreased CoQ9/CoQ10 ratio in animals fed on the PUFAn-6-rich plus CoQ10 diet could also influence longevity is also discussed.

Coenzyme Q and the regulation of intracellular steady-state levels of superoxide in HL-60 cells.

The present work was set to study how CoQ concentrations affected steady-state levels of superoxide in a cellular model of partial CoQ(10) deficiency in cultured human myeloid leukemia HL-60 cells. Culturing HL-60 cells in the presence of p-aminobenzoate, a competitive inhibitor of polyprenyl-4-hydroxybenzoate transferase (Coq2p), produced a significant decrease of CoQ(10) levels without affecting cell viability. Concomitant decreases in CoQ-dependent electron transport activity and mitochondrial membrane potential were observed under these conditions. Intracellular superoxide was significantly elevated in cells treated with p-aminobenzoate, both under serum-containing and serum-free conditions, and this effect was reversed by exogenous CoQ(10). A slight increase of superoxide was also observed in CoQ(10)-supplemented cells in the absence of serum. Our results support a requirement for CoQ(10) to control superoxide levels in HL-60 cells. The importance of extramitochondrial sources of superoxide in cells with impaired CoQ(10) biosynthesis is discussed.

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells.

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQOI, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b5 reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q0 through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b5 reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b5 reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.